Dan Putnam, forage specialist at the University of California – Davis, recommends the following sampling procedures for detection of a GE trait in alfalfa:
Identify a single lot
This is a key first step to proper hay sampling, and one frequently ignored. Normally, a hay lot should be identified, which is a single cutting, a single field and variety, and generally be less than 200 tons. For exports, a hay lot can be considered a single container or group of containers. However, in principle, the lot should consist of hay originating from a single field, single cutting and be as uniform as possible. For exports, identification of grower(s) and source of hay is important.
When to sample
For detection of GE traits for export, sampling can occur in defined lots before double compression. Care should be taken to keep identification of these lots throughout the repackaging process, and quality-assurance processes by exporters (including spot-checking compressed bales) may assist confirming the determination of non-GE status in the final double-compressed product. Sampling can also occur after double compression to confirm these results; however, different coring devices may be necessary.
Choose a sharp, well-designed coring device
We generally recommend a sharp coring device three-eighths to three-fourths of an inch in diameter, approximately 16 to 24 inches in length, which is capable of penetrating a bale 12 to14 inches. Do not use flakes or grab samples. The probe should be capable of penetration (whether single- or double-compressed) and must fairly represent the leaf stem ratio in a cross section.
Probes larger than three-fourths of an inch are acceptable for GE testing, but may inadvertently create samples that are too large to be handled by a laboratory, or make the sampler stop before the prescribed number of samples is obtained. Thus, smaller diameter probes are preferred, as long as they are capable of obtaining a representative cross section of stem and leaf. The principle is to sample as many stems as possible, not maximize the size of the sample.
Larger diameter probes are not likely to sample a significantly greater number of stems than a smaller diameter probe at a depth of 12 to 14 inches. A range of probe-tip designs have been successfully used, from serrated to non-serrated tips. It is probably most important that the tip be sharp (and maintained sharp) and creates a clean cut across a cross section of hay and does not heat during the sampling process. Not all probes meet these criteria.
Note: Only a few probes can be practically used for double-compressed hay. Gas powered drills utilizing a spiral-assist probe (like the Star-Quality Sampler, Edmonton, AB) have been shown to work, as have some punch-type probes, which require strength. In both cases, tips must be sharp. We have found serrated-type probes (e.g., Penn State probe) that heat too fast to be practical.
Take enough cores
For GE sampling, 30 cores composited into a single sample is likely to provide a sample that represents low-level presence (LLP), if present, at about 0.1 percent, if the number of stems in each probe is above about 250 per probe. In practice we have found the range of stems to be from about 100 to 400 per probe.
This is greater than the 20 probes recommended for forage quality testing – since in this case we are looking for low-level presence, not the average quality of the sample. This is the same recommendation for larger (e.g., 1 ton) or smaller packages, as long as these groups of bales fit the definition of a ‘lot’ (see ‘identify a single lot’ above). The key aspect of sampling hay bales is the number of samples composited, not necessarily the mass of the sample collected, as long as the mass is sufficient to represent discrete stems in each probed sample.
Sample at random
These 30 cores should be taken without bias. Ideally, the sampler should walk around the stack as much as possible and sample bales at random, and both ends of bales should be sampled. This is sometimes difficult since not all of the bales are available to the sampler. (They may be against the walls of a barn, present only on one face of a container or stacked too high for practical sampling.) However, the sampler should make every attempt to sample in a random fashion. This means not to bias either for or against any bales in the stack.
For example, the sampler may walk 15 steps, sample, walk 20 steps, sample, walk five steps, sample, while walking around the stack, trying to represent all areas of the stack. Don’t avoid or choose bales because they look especially bad or good. If 20 cores are taken, they won’t make much difference anyway. Avoiding or choosing bales introduces bias. Note for bales in a container: 25 to 30 cores can be taken from the face presented to the sampler; even multiple samples can be taken from the same bale, since there is often as much variation within bales as between bales. The key is to make sure that 25 to 30 cores are taken, and that each core is at least 30 inches (0.7 meter) away from other sample cores to assure that the same plants are not sampled.
Use proper technique
Sample butt ends of hay bales near the center, in the compacted area between strings or wires, not near the edge. The butt ends of the bales should have the stems of the product perpendicular to the probe. The probe should be inserted at a 90-degree angle, 12 to 14 inches deep. Sample multiple bales if possible, but if sampling the same bales, assure that samples are at least 30 inches apart. The sides or the top of the bale should not be sampled, since these cores will only represent one flake from a single area of the field, and the stems will not be perpendicular to the probe. With round bales, samples should be taken towards the middle of the bale, on an angle directly towards the center of the bale.
Sample amount: Not too big, not too small
Sampling should be done so that about one-half pound (226 g) to 1 pound (454 g) of sample is produced, taking a minimum of 30 cores. For LLP testing, larger is better – but make sure the laboratory grinds the entire sample. Samples too small do not fairly represent the full range of variation in the hay lot. Very large samples (common with large length or diameter probes) are excellent at representing the hay but have practical disadvantages. Large samples cannot be easily ground by the labs. The sampler should ensure that the entire sample is ground by the lab. Only work with labs that are willing to grind the entire sample. Also assure that you are providing a reasonable weight sample, so that it can be practically handled by the lab.
Handle samples correctly
Seal a composite 30-core sample in a well-sealed plastic bag and protect from heat. Double bagging is beneficial, especially for dry matter measurements. Deliver to the lab as soon as possible. Do not allow samples to be exposed to excess sun (e.g., in the cab of a pickup truck). Refrigeration of hay samples is helpful; however, dry hay samples (about 90 percent dry matter) are considered fairly stable. Note: This is more important for quality analysis than for GE analysis.
Choose a lab with established methods
For forage quality testing, we recommend labs that participate in the National Forage Testing Association proficiency certification program. For polymerase chain reaction testing, consult recommendations of the regulatory agency for labs that have been approved.
First published in the 2014 California Alfalfa, Forage & Grains Symposium proceedings.
PHOTO
A worker at Aldahra ACX Global in Wilmington, California, stacks hay in preparation for export from the Port of Long Beach. Photo by Lynn Jaynes.
